Anal. Chem. 1999, 71, 115-118
Membrane Supports as the Stationary Phase in
High-Performance Immunoaffinity Chromatography
Dongmei Zhou, Hanfa Zou,* Jianyi Ni, Li Yang, Lingyun Jia, Qiang Zhang, and Yukui Zhang
National Chromatographic R & A Center, Dalian Institute of Chemical Physics, The Chinese Academy of Sciences,
Dalian 116011, China
The membrane with a composite of cellulose grafted with
acrylic polymers formed by polymerizing a glycidyl methacrylate in the presence of dispersed cellulose fiber was
prepared as the stationary phase; the column (40 × 4 mm
i.d.) which was compatible with the HPLC instrument was
packed with pieces of the cut membrane. Protein A and
human IgG were immobilized on the membrane stationary
phase. The column based on the membrane support
provided us good reproducibility, high efficiency, and low
back pressure. High-performance immunoaffinity chromatographic analysis of human IgG in serum and polyclonal antibody to human IgG raised in goat was performed within 2.5 min. The fast-speed immunoaffinity
analysis was developed by increasing the flow rate of the
mobile phase and decreasing the duration time for the
switch of the mobile phase; an operation for immunoaffinity analysis of human IgG could be finished within
30 s.
High-performance liquid chromatography (HPLC) revolutionized analytical chemistry by facilitating very rapid and efficient
separations and the detection and determination of the components of virtually any mixture. The separation, isolation, and
purification of biopolymers is very important for their effective
application. The analytical and preparative HPLC separations of
individual biological macromolecules from their mixtures with
both low- and high-molecular-mass compounds has been reviewed
several times.1-3 At present, most chromatographic separations
are carried out in column packed almost exclusively with beadshaped particles,4 as the technology of bead preparation has been
known for more than two decades. Recently, a new technique
called “perfusion chromatography” was invented,5-7 which involves
the flow of liquid through a porous chromatographic particle for
(1) Frenz, J.; Horvath, Cs. In High-Performance Liquid Chromatography: Advances
and Perspectives; Horvath, Cs., Ed.; Academic Press: New York, 1988; Vol.
5, p 211.
(2) Gooding, K. M.; Reginer, F. E. Eds. HPLC of Biological Molecules, Methods
and Applications; Marcel Dekker: New York, 1990.
(3) Mant, C. T.; Hodges, R. S. Eds. High-Performance Liquid Chromatography
of Peptides and Proteins: Separation, Analysis and Conformation; CRC Press:
Boca Raton, FL, 1991.
(4) Unger, K. K., Ed. Packings and Stationary Phases in Chromatographic
Techniques; Marcel Dekker: New York, 1990.
(5) Afyan, N. B.; Gordon, N.; Maszaroff, I.; Varady, L.; Fulton, S. P.; Yang, Y.;
Regnier, F. E. J. Chromatogr. 1990, 519, 1.
(6) Afeyan, N. B.; Fulton, S. P.; Regnier, F. E. J. Chromatogr. 1991, 544, 267.
(7) Fulton, S. P.; Afeyan, N. B.; Gordon, N. F.; Regnier, F. E. J. Chromatogr.
1992, 547, 452.
10.1021/ac980613i CCC: $18.00
Published on Web 12/03/1998
© 1998 American Chemical Society
reducing resistance to stagnant mobile-phase mass transfer
without sacrificing adsorbent capacity or necessitating extremely
high-pressure operation. Many applications of perfusion chromatography to separations of biopolymers have been reported.8-10
Traditional membranes were introduced into affinity chromatography in 1988.11-13 Ion-exchange cellulose membranes stacked
in a cartridge also gave good results in the separation and
purification of proteins.14,15 The recently introduced high-performance membrane chromatography (HPMC) combines the advantages of both membrane technology (simple scale-up, lowpressure drop across a membrane, and low cost) and column
chromatography (high selectivity and efficiency of separation, high
loading capacity).16-19 The detailed studies of individual effects
of variable parameters in ion-exchange, hydrophobic interaction,
and reversed-phase protein separations confirmed that HPMC
obeys the rules typical of HPLC.20 With comparison of HPMC to
perfusion chromatography, both perfusion chromatography and
HPMC can be easily scale-up for purification of biopolymers, but
the cost of perfusion chromatography is much higher than that
of HPMC. In this work, high-performance immunoaffinity chromatographic columns with immobilization of ligands of protein A
and human IgG on the membrane support have been prepared;
analyses of human IgG in serum and polyclonal antibody to human
IgG raised in goat serum have been performed.
(8) Fulton, S. P.; Meys, M.; Varady, L.; Jansen, R.; Afeyan, N. B. Biochromatography 1991, 11, 226.
(9) Zou, H. F.; Zhang, Y. K.; Lu, P. C.; Krull, I. S. Biomed. Chromatogr. 1996,
10, 78.
(10) Zou, H. F.; Zhang, Y. K.; Lu, P. C.; Krull, I. S. Biomed. Chromatogr. 1996,
10, 122.
(11) Huang, S. H.; Roy, S.; Hou, K. C.; Tsao, G. T. Biotechnol. Prog. 1988, 4,
159.
(12) Hou, K. C.; Zaniewski, R. J. Chromatogr. 1990, 525, 159.
(13) Langlotz, P.; Kroner, K. H. J. Chromatogr. 1992, 591, 107.
(14) Tan, L. U. L.; Yu, E. K. C.; Luis-seize, G. W.; Saddler, J. N. Biotechnol. Bioeng.
1987, 30, 96.
(15) Wang, H.; Li, T.; Zou, H.; Zhang, Y.; Chao, J.; Chao, L. Biomed. Chromatogr.
1996, 10, 139.
(16) Tennikova, T. B.; Bleha, M.; Sevc, F.; Almazova, T. V.; Belenkii, B. G. J.
Chromatogr. 1991, 555, 59.
(17) Abou-Rebyeh, H.; Korber, F.; Schubert-Rehberg, K.; Reusch, J.; Josic, Dj. J.
Chromatogr. 1991, 566, 341.
(18) Josic, Dj.; Reusch, J.; Loster, K.; Baum, O.; Reutter, W. J. Chromatogr. 1992,
590, 59.
(19) Josic, Dj.; Lim, Y. P.; Strancar, A.; Reutter, W. J. Chromatogr. 1994, 662,
217.
(20) Tennikova, T. B.; Sevc, F. J. Chromatogr. 1993, 646, 279.
Analytical Chemistry, Vol. 71, No. 1, January 1, 1999 115
glutaraldehyde was removed by washing the column with 40 mL
of borate buffer. This chemical reaction can be described by
following equations:
(2)
Figure 1. Chemical structure of GMA-cellulose composite membrane.
EXPERIMENTAL SECTION
Preparation of Membrane Media. The chromatographic
solid matrix is a composite of cellulose grafted with acrylic
polymers formed by polymerizing a glycidyl methacrylate in the
presence of dispersed cellulose fiber, followed by an in situ
covalent binding of the acrylic polymer to the cellulose as
described by Hou et al.,21 which is named arcylic membrane here.
The reaction processes are shown in Figure 1. The bicomponent
fiber thus formed consisted of a cellulosic core as the mechanical
support and the acrylic sheath as a chemical functional group
carrier. The composite fiber can be further derivatized to the
required functional groups. The media with amino groups were
prepared by reacting the glycidyl groups grafted on the fiber
surface with hexyldiamine monomers at 80 °C for an additional
hour; it is named amino membrane here.
Preparation of Immunoaffinity Column. The composite
fiber carrying specific functional groups was then fabricated in
paper form by a conventional paper-forming machine. Pieces of
membrane with 4.0-mm diameter were cut off; the column (40 ×
4 mm i.d.), which was compatible with the HPLC instrument, was
packed with the cut membrane pieces until full. Two approaches
were used for the immobilization of protein A on the acrylic and
amino membranes, respectively. (1) The aldehyde groups on the
acrylic membrane were generated directly from the glycidyl
groups by acid hydrolysis with HCl at pH 0.6 for 6 h to form vicinal
hydroxyl groups, followed by periodate oxidation with NaIO4 (12%) for 30 min. Those chemical reactions can be described by
following equation:
(1)
M in eq 1 is the membrane. (2) Aldehyde groups on the amino
membrane were generated from the amino matrixes by recirculating 0.1 mol/L sodium borate and HCl buffer, pH 8.2, containing
0.25% glutaraldehyde for 2 h at room temperature. Excess
(21) Hou, K. C.; Zanieweski, R.; Roy, S. Biotechnol. Appl. Biochem. 1991, 13,
257.
116
Analytical Chemistry, Vol. 71, No. 1, January 1, 1999
The coupling of protein A with the aldehyde group-generated
membranes was performed by recirculating the solutions of 4 mg/
mL protein A in 0.1 mol/L borate buffer, pH 8.2, in the presence
of 0.025 mol/L NaCNBH3 for 15 h at room temperature. The
uncoupled ligand was removed by rinsing the column with buffer
solution until the ligand is no longer detectable in the eluent. The
excess active groups were deactivated by recirculating 1.0% glycine
ethyl ester hydrochloride in 0.1 mol/L sodium phosphate, pH 6.5,
containing the reduction agent for 4 h. The immunoaffinity column
was washed extensively and then equilibrated in the loading buffer
for the evaluation and analysis of human IgG.
The immunoaffinity column by immobilization of human IgG
was also prepared by the same method reported above and used
for analysis of goat anti-HIgG.
Chromatography. The HPLC system used was two Waters
510 pumps (Waters, Milford, CT) controlled by a WDL-95
workstation (National Chromatographic R&A Center, Dalian,
China). Eluates were detected at wavelength of 280 nm by a
Spectra-200 UV detector (Spectra-Physics, San Jose, CA). Human
IgG and bovine serum albumin (BSA) were purchased from Sigma
Chemical Co. (St. Louis, MO). Human serum and a crude powder
of goat-polyclonal antibody to human IgG (goat anti-HIgG) were
obtained from the Dalian Hospital (Dalian, China) and Tianjin
Central Hospital (Tianjin, China), respectively. All other chemicals
were analytical-reagent grade, and the solutions were made in
double-distilled water. The loading buffer for HPIAC was 10
mmol/L phosphate buffer containing 0.15 mol/L NaCl, and the
pH value of loading buffer was adjusted to 7.2 with NaOH. The
elution buffer was 0.15 mol/L of NaCl in water adjusted to pH 2.6
with HCl.
RESULTS AND DISCUSSION
Figures 2 and 3 showed the chromatograms to test the
nonspecific adsorption of proteins on the protein A columns based
on the coupling of protein A on membrane supports through the
chemical reactions shown as in eqs 1 and 2, respectively. Curve
3 in Figures 2 and 3 represents the real chromatograms, the
difference between curves 1 and 2 shown in Figures 2 and 3,
where curves 1 and 2 were obtained by injections of 20 µL of
loading buffer containing 100 µg of BSA and only loading buffer
itself, respectively. It can be seen that there was a small peak
observed in Figure 3 when the eluent was changed from the
loading buffer to the elution buffer, which was caused by the
nonspecific adsorption of BAS on the protein A column due to
the long coupling arm prepared by the chemical reaction of eq 2.
But no such a nonspecific adsorption was observed for the protein
A column on the stationary phase prepared by the chemical
reactions of eq 1, because no peak was observed when the eluent
was changed from the loading buffer to the elution buffer. All
a
Figure 2. Chromatograms of BSA solution on the protein A column
by its immobilization on the membrane through the chemical reactions
shown in eq 1. Experimental conditions: 20 µL of sample solution
was injected with loading buffer as the eluent, and 2 min after injection
of sample solution, the elution buffer was used as the eluent. Flow
rate 1.0 mL/min; detection wavelength 280 nm. Curves: (1) loading
buffer containing 100 µg of BSA; (2) loading buffer; (3) difference
between curves 1 and 2.
b
Figure 4. Chromatographic analysis of human IgG on the protein
A column. Experimental conditions: (a) 20 µL of human IgG solution
(2.5 mg/mL) and (b) 10-fold diluted solution of human serum were
injected. Other experimental conditions were the same as in Figure
2. Chromatographic peaks: (1) nonretained solutes; (2) human IgG.
Figure 3. Chromatograms of BSA solution on the protein A column
by its immobilization on the membrane through the chemical reactions
shown in eq 2. Experimental conditions: 20 µL of sample solution
was injected with loading buffer as the eluent, and 2.3 min after
injection of sample solution, the elution buffer was used as the eluent.
Other experimental conditions were the same as in Figure 2.
Curves: (1) loading buffer containing 100 µg BSA; (2) loading buffer;
(3) difference between curves 1 and 2.
experiments below were performed on the protein A column based
on the membrane support prepared by the chemical reactions of
eq 1.
The pressure drop of the column was determined by changing
the flow rate of the mobile phase, and it was observed that the
column packed with the membrane support has a very low
pressure drop, which is important for high-speed analysis in highperformance immunoaffinity chromatography. For example, the
pressure drop of the column was only about 4 MPa even at a flow
rate of 3.0 mL/min.
Figure 4 showed the typical chromatograms of human IgG
standard sample and diluted human serum on the protein A
column, which indicated that an immunoaffinity analysis of the
human IgG sample could be finished within 2.5 min at a flow rate
of 1.0 mL/min, and the peak width at baseline for the retained
human IgG is less than 0.2 min. The reproducibility of the column
was evaluated by five injections of 10-fold diluted human serum,
and it was observed that the protein A immunoaffinity column
based on membrane support gave very good reproducibility of
peak areas with RSD less than 1.0% for the nonretained impurities
and the retained human IgG.
The calibration curve for quantitation of human IgG on the
protein A column by injection of the standard human IgG samples
was determined, and a good linearity of the peak area of retained
human IgG versus the injection amount of human IgG in the range
3-75 µg was obtained with a regression coefficient higher than
0.999. From the average peak areas of the retained IgG measured,
the amount of human IgG in serum can be calculated by the
calibration curve as 14 mg/mL.
Fast-speed analysis of human IgG on the protein A immunoaffinity column with membrane support was developed by increasing the flow rate of the mobile phase and decreasing the duration
time for switching of the mobile phase. Figure 5 showed typical
chromatograms for injection of the standard sample and the 10fold diluted human serum. It can be seen that one operation for
the immunoaffinity analysis of human IgG can be finished in even
less than 30 s, and the peak width at baseline for the retained
human IgG was about 5 s. The amount of human IgG in serum
can be quantitated as 13.6 mg/mL by comparison of peak areas
for the retained human IgG in the standard sample and diluted
human serum. This result means that the amount of human IgG
in serum measured by fast-speed analysis is about 3% lower than
that measured by the utilization of a calibration curve measured
at a flow rate of 1 mL/min, which may be caused from the
impurities that existed in the standard sample of human IgG
shown in peak 1 in Figure 5a.
Analytical Chemistry, Vol. 71, No. 1, January 1, 1999
117
a
a
b
b
Figure 5. Fast-speed analysis of human IgG on the protein A
column. Experimental conditions: (a) 20 µL of human IgG solution
(2.5 mg/mL) and (b) 10-fold diluted solution of human serum were
injected, and 20 s after injection of sample solution, the elution buffer
was used as the eluent. Flow rate 3.0 mL/min; other experimental
conditions were the same as in Figure 4. Chromatographic peaks:
(1) nonretained solutes; (2) human IgG.
Furthermore, the immunoaffinity analysis of goat-polyclonal
antibody to human IgG (goat anti-HIgG) in crude powder was
carried out on the protein A and human IgG columns; the latter,
the human IgG, was immobilized on the membrane support. The
obtained chromatograms were shown in Figure 6. It can be seen
that the peak for the retained goat-anti HIgG on the protein A
column is much smaller than that on the human IgG column,
which means that the immunoaffinity interaction between protein
A and goat anti-HIgG is much weaker than that between HIgG
and goat anti-HIgG. The reproducibility of the HIgG column for
analysis of goat anti-HIgG was tested by five injections of goat
anti-HIgG solution, made from the crude powder dissolved in the
loading buffer; it was observed that the HIgG column also gave
quite good reproducibility with a RSD of 3.2% for the peak areas
of retained goat anti-HIgG. It is assumed that the goat anti-HIgG
has the same UV absorpitvity as the human IgG at 280 nm; then
we can roughly estimate that the amount of goat anti-HIgG in
crude powder is about 105 µg/mg.
CONCLUSION
Columns with membrane support as the stationary phases in
high-performance immunoaffinity chromatography have been
118 Analytical Chemistry, Vol. 71, No. 1, January 1, 1999
Figure 6. Chromatograms of goat anti-HIgG solution on the (a)
protein A and (b) HIgG columns. Experimental conditions: 20 µL of
solution by dissolving crude powder of goat-anti HIgG in loading buffer
(5 mg/mL) was injected. Other experimental conditions were the same
as in Figure 3. Chromatographic peaks: (1) nonretained solutes; (2)
goat anti-HIgG.
developed. The column developed provided us good reproducibility, high efficiency, and low back pressure. High-performance
immunoaffinity chromatographic analysis of human IgG in serum
and polyclonal antibody to human IgG raised in goat were
performed on the protein A and human IgG columns, respectively;
usually an immunoaffinity analysis of the human IgG and goat
anti-polyclonal antibody could be finished within 2.5 min at a flow
rate of 1.0 mL/min. Furthermore, the fast-speed analysis of the
human IgG within 30 s on the immunoaffinity column was
developed by increasing the flow rate of mobile phase and
decreasing the duration time for switching of the mobile phase.
ACKNOWLEDGMENT
Financial support from the National Natural Science Foundation of China (No. 29635010) is gratefully acknowledged. H.Z. is
recipient of the excellent young scientist award from the National
Natural Science Foundation of China (No. 29725512).
Received for review June 4, 1998. Accepted October 15,
1998.
AC980613I